Phospholipid-based inhalation system

ABSTRACT

The present invention includes a method for delivering medications deeper into the lungs and to the medications&#39; pulmonary targets, which include bronchioles and alveoli. A first particularly preferred embodiment of the invention describes the use of two steps for delivery of a medication. In a first step, an aerosolized therapeutic composition or medication is administered into a patient&#39;s respiratory tract, wherein the patient may be any animal or human subject. Following the first step, an aerosolized surfactant is administered into the patient&#39;s respiratory tract that facilitates delivery of the aerosolized medication of the first step to the medication&#39;s pulmonary target. Another embodiment of the present invention contemplates an apparatus for the delivery of an aerosolized surfactant used that facilitates delivery of previously inhaled aerosolized medication.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 11/977,932, filed Oct. 26, 2007, now U.S. Pat. No.8,206,687, issue date Jun. 26, 2012, which claims priority to U.S.Provisional Patent Application Ser. No. 60/854,596 filed Oct. 26, 2006,from which this application also claims priority, each of theabove-reference applications is hereby incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention generally relates to the field of drug delivery. Moreparticularly, this invention is directed to inhaled medications (forexample medications delivered through pressurized metered dose inhalers(“pMDIs”) or other inhalers) and the delivery of medications toconducting airways and alveoli in a respiratory system.

2. Background of the Invention

Inhaled medications are commonly used to target drugs to the lungs inthe treatment and prevention of various medical conditions. A. Steimer,E. Haltner, & C. M. Lehr, Cell Culture Models of the Respiratory TractRelevant to Pulmonary Drug Delivery, 18 J. Aerosol Med. 137 (2005); R.Dalby & J. Suman, Inhalation Therapy: Technological Milestones inAsthma, 55 Adv. Drug. Del. Rev. 779 (2003). Drugs administered throughthe pulmonary route either act locally in the lungs or enter thesystemic circulation following dissolution and absorption. Numerousparticle and device engineering approaches have been attempted toincorporate drugs into small particles or make small pure drug particlesfor delivery to the most desirable lung locations. Such approachesinclude modifications to nebulizers, pressurized metered dose inhalers(pMDIs), active or passive dry powder inhalers (DPIs), or changes to thenature of the particles themselves. The ultimate objectives of particleand device engineering are to generate small slow moving particles withfavorable aerodynamic properties. S. J. Farr, S. J. Warren, P. Lloyd, J.K. Okikawa, J. A. Schuster, A. M. Rowe, R. M. Rubsamen & G. Taylor,Comparison of in Vitro and in Vivo Efficiencies of a Novel Unit-DoseLiquid Aerosol Generator and a Pressurized Metered Dose Inhaler, 198Int. J. Pharm. 63 (2000); VIII R. W. Niven, Respiratory Drug Delivery,Powders and Processing: Deagglomerating of a Dose of Patents andPublications 257-266 (R. N. Dalby, P. Byron, J. Peart, & S. Farr eds.,DHI, Rayleigh 2002); K. R. Chapman, L. Love, & H. Brubaker, A Comparisonof Breath-Actuated and Conventional Metered-Dose Inhaler InhalationTechniques in Elderly Subjects, 104 Chest. 1332 (1993).

The fraction of drug delivered to the bronchial tree may be cleared bymucociliary transport and absorption through the airway epithelium intothe systemic circulation. Thus, after initial deposition, drug particlesdo not migrate deeper into the lung. The opposite occurs: once particlesencounter the fluid lining of the lung; they are either absorbed ortransported to the larger airways of the lung by lung clearancemechanisms. Drug reaching the target region (which may be conductingairways or alveoli) of the lung following pulmonary inhalation(expressed as bioavailability or a deposition fraction) is oftenestimated at less than 10%. VIII M. Sakagami, Respiratory Drug Delivery,Pulmonary Insulin: a Critical Review of Its Biopharmaceutics 69-78 (R.N. Dalby, P. Byron, J. Peart, & S. Farr eds., DHI, Rayleigh 2002).

Following premature births, structurally immature andsurfactant-deficient lungs containing reduced levels of pulmonaryphospholipids are sometimes treated with natural and synthetic exogenoussurfactants (treatment of Respiratory Distress Syndrome RDS). G. K.Suresh & R. F. Soll, Lung Surfactants for Neonatal Respiratory Syndrome:Animal Derived or Synthetic Agents, 4 Pediatr. Drugs. 485 (2002). Theseexogenous surfactants are complex colloidal dispersions composedprimarily of phospholipids. They may contain additional components suchas fatty acids, triglycerides and spreading agents. The dose ofsurfactant is relatively high and is administered to premature infantsaffected with RDS via endotracheal or intratracheal instillation whereinthe surfactant is dripped directly into the bronchioles.

After instillation, the surfactant is distributed throughout the airwaysand the bolus advances distally while coating the airway walls with athin layer of surfactant. F. F. Espinosa & R. D. Kamm, Bolus DispersalThrough Lungs in Surfactant Replacement Therapy, 86 J. Appl. Physiol.391 (1999). The thickness of the coat of surfactant is dependent onsurfactant concentration, viscosity and surface tension. In addition, a“reservoir” of surfactant remains in the larger airways as thesurfactant expands into the lungs. Surface tension gradients drawexogenous surfactant distally to high surface tension locations therebyallowing surfactant to reach the alveoli.

FDA approval and continuous commercial availability of exogenoussurfactants and their use in critically ill neonatal patients confirmsthe safety of phospholipid administration to the human respiratorytract. Their mode of administration (tracheal instillation) and site ofaction (alveolar spaces) confirms that the active components of thesesurfactant mixtures successfully spread from the trachea to the alveolito exert their beneficial effect. R. J. Rodriguez, Management ofRespiratory Distress Syndrome: An Update, 48 Respir. Care. 279 (2003).

SUMMARY OF THE INVENTION

The present invention provides a method for delivering deposited drugparticles or droplets containing dissolved drug deeper into therespiratory tract or human or animal subjects in front of the spreadingsurfactant layer, thus increasing the fraction of drug that reaches thedesirable targets in the bronchioles and alveolar spaces of the lung.One particularly preferred embodiment of the invention is a method fordelivering medications or particles to pulmonary targets where a firststep of administering an aerosolized (inhaled) medication into apatient's respiratory tract is followed by a second step ofadministering an aerosolized (inhaled) surfactant into the patient'srespiratory tract. The second step facilitates deeper lung penetrationof the aerosolized medication administered in the first step by pushingthe medication deeper into the lungs and closer to the medication'spulmonary target. Consequently, the patient inhales his or hermedication from an existing inhaler (such as a pMDI) containing thedesired medication such that drug (or drug in droplets) deposits on theluminal surface. The location of deposition of the particles depends oninhaler characteristics and patient technique. The patient immediatelyinhales a dose of surfactant from a second inhaler containing surfactantparticles (e.g., phospholipid molecules) or droplets larger than thedrug particles. The larger surfactant particles deposit higher in theairway and, as the surfactant dissolves and spreads deeper into therespiratory tract, drug particles are pushed deeper into the lungs aheadof the expanding surfactant layer.

A second aspect of the present invention contemplates an apparatus fordelivery of aerosolized surfactants to be administered after initialinhalation of an aerosolized medication, comprising a pressurized meterdose inhaler, DPI or nebulizer formulation. For example, the pressurizedmeter dose inhaler formulation would comprise a surfactant thatfacilitates delivery of the aerosolized medication and a propellant.

A further aspect of the present invention includes a method forselecting a surfactant formulation that facilitates the delivery of anaerosolized medication, comprising determining an aerodynamic particlesize distribution for a surfactant formulation. The aerodynamic particlesize distribution is determined using a method wherein a surfactantformulation is delivered into a dome connected to an impactor. Thesurfactant that deposits on the impactor is then collected and itsconcentration measured.

Yet a further aspect of the present invention comprises a method ofselecting molecules to act as surfactants for delivery of aerosolizedmedications, comprising monitoring migration of latex beads placed ontissue cells after application of a surfactant formulation.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other features, aspects, and advantages of the presentinvention are considered in more detail, in relation to the followingdescription of embodiments thereof shown in the accompanying drawings,in which:

FIG. 1 provides a graphical overview of a method of deliveringmedications to the lungs.

FIG. 2 describes a modified Andersen Cascade Impactor “ACI” system, the“dome” method, used to determine the aerodynamic particle size ofsurfactant formulation. The “dome” 201 has a capacity of approximately15 L and is attached on top of the ACI 204. Aerodynamic size ofaerosolized particles from a pMDI formulation 207 is measuredconsecutively.

FIG. 3 displays a sample of a microgrid used to count and monitormovement of latex beads as a tool for identifying appropriate pMDIformulations. Location 7 307 is near the point of surfactant applicationand location A 310 is furthest away within a counting lane 313 (shadedregion). Each small grid square 301 represents 55 μm².

FIG. 4 provides a representation of a V-adaptor used to applyphospholipids to the microgrid shown on FIG. 3: V-piece 401, pMDI spray404, Slit/opening 407 for spray passage and Coverslip 410.

FIG. 5 displays an example of results that can be obtained using thebead migration procedure. (A) Panel (I) shows a 4.5 μm bead distributionbefore (Initial) application of phosphatidylcholine (PTC) onrepresentative grids at location A 310 (1650 μm grid distance) andlocation 7 307 (110 μm grid distance). Panel (II) shows beaddistribution after (Final) application of PTC on representative grids atlocation A 310 and location 7 307. (B) Panel (III) shows a 4.5 μm beaddistribution before (Initial) application of water (control) onrepresentative grids at location A 310 and location 7 307. Panel (IV)shows bead distribution after (Final) application of water onrepresentative grids at location A 310 and location 7 307. The plotsrepresent bead count ratio (Final:Initial) as a function of griddistance. These show significant migration of latex beads on applicationof PTC as the surfactant. However, no such migration was observed whenwater was used as the control. Vertical bars represent mean±S.D. oftriplicate experiments. Similar pictures and graphs can be obtainedusing this method with any prospective surfactant formulation.

FIG. 6 demonstrates results of the procedure as shown in FIG. 5utilizing a different surfactant. Particle migration on A549 cellsurface on application of Survanta® (Abbott Laboratories, Corp., AbbottPark, Ill. 60064) as monitored using CELLocate microgrid system, plottedas a function of bead count ratio (Final:Initial). Phospholipid wasapplied on the 55 μm grid distance end (location 7 307) and latex beadsmigrated away towards the 1760 μm end. Data represent mean±S.D. oftriplicate experiments.

FIG. 7 describes another modification of the bead migration proceduredescribed in FIG. 5, where the effect of the surfactant concentration onmigration is quantified. The latex bead count ratio is plotted as afunction of grouped segment position demonstrating the concentrationeffect of CFC-12 pMDI-delivered Survanta® on particle migration acrossan alveolar cell surface, measured using the semi-automated system.pMDI-delivered Survanta® was applied near the 220 μm segment location.pMDI-delivered Survanta® 0.6 mg/mL▪=72 μg of administered phospholipid;pMDI-delivered Survanta® 1.3 mg/mL

=163 μg of administered phospholipid; pMDI-delivered Survanta® 1.8mg/mL□=221 μg of administered phospholipid; pMDI-delivered Survanta® 6.2mg/mL

=786 μg of administered phospholipids. Data represent mean±S.D. oftriplicate observations with each pMDI actuated twice per observation.

FIG. 8 provides an example of latex bead count ratio plotted as afunction of grouped segment position demonstrating the effect of HFA-227pMDI-delivered Survanta®, at a concentration of 1.5 mg/mL, on particlemigration across an alveolar cell surface. The plot shows the effect oftwo different formulations containing 10% w/w ▪ and 20% w/w

of ethanol. Data represent mean±S.D. of triplicate observations witheach pMDI actuated twice per observation.

FIG. 9 describes surface tension of six selected CFC-12 containingSurvanta® pMDIs administered onto Hank's balanced salt solution (surfacetension 68.41±0.71 mN/m) performed using Du Noüy Tensiometer at 25° C.;vertical bars represent mean±S.D.

DETAILED DESCRIPTION

The invention summarized above may be better understood by referring tothe following description, which should be read in conjunction with theaccompanying drawings and claims. This description of an embodiment, setout below to enable one to build and use an implementation of theinvention, is not intended to limit the invention, but to serve as aparticular example thereof. Those skilled in the art should appreciatethat they may readily use the conception and specific embodimentsdisclosed as a basis for modifying or designing other methods andsystems for carrying out the same purposes of the present invention.Those skilled in the art should also realize that such equivalentassemblies do not depart from the spirit and scope of the invention inits broadest form.

The present invention includes a method for delivering medicationsdeeper into the lungs and to the medications' pulmonary targets, whichinclude bronchioles and alveoli. It is contemplated that a medicationincludes any particle, molecule or composition administered to asubject, human or animal, to achieve any desired result. A firstparticularly preferred embodiment of the invention describes the use oftwo steps for delivery of a medication. In a first step, an aerosolizedmedication is administered into a patient's respiratory tract, whereinthe patient may be any animal or human subject. Following the firststep, an aerosolized surfactant is administered into the patient'srespiratory tract that facilitates delivery of the aerosolizedmedication of the first step to the medication's pulmonary target. Forexample, as shown in FIG. 1, the patient 101 inhales his or hermedication from a container 104, such as a pMDI. It is contemplatedthat, throughout this specification, the term “particle” or “particles”includes mixtures containing the medication or surfactant, droplets inwhich the medications or surfactants have been dissolved, solidmedication or surfactant particles, and any other compositioncontemplated by one skilled in the art to contain the medication orsurfactant being administered to the patient. The particles or dropletsdeposit on the luminal surface 113. The location for deposit of theparticles depends on the inhaler characteristics and patient technique.The patient then inhales a dose of the aerosolized surfactant from asecond inhaler 107. The surfactant contains phospholipid particles thatpush the medication to its pulmonary target. The phospholipid molecules,packed in aerosolized particles larger than those in which themedication is delivered, deposit at a higher location in the airway 110and slide down the oropharyngeal region 113 creating a gradient andpushing the smaller particles, containing the medications, deeper intothe lungs 116. The same effect can be obtained through the use of atandem inhaler apparatus, or any other application that allows thesmaller particles containing the medications to be delivered first,followed by the larger particles containing a surfactant that includesphospholipid “chaser” molecules.

The surfactant to be used in delivering medications can be packaged in acontainer 104 (such as a pMDI, nebulizer, dry powder inhaler, or anyother aerosolized delivery mechanism) comprising a propellant and asurfactant formulation that facilitates delivery of the medication. Forexample, surfactant formulations to be used in the second pMDI underthis method may include various volumes of a surfactant, e.g. PTC orSurvanta®, containing a volume of phospholipids, e.g., 25 mg/mL,packaged into glass pMDI canisters and freeze-dried at an appropriatetemperature, e.g., −80° C., to remove water. The freeze-dried productcan be dispersed using anhydrous ethanol in a desired volume, e.g., 1mL. The pMDIs are then crimped with appropriate nitrile valves, such as63 μL (Bespak plc, Milton Keynes, UK), and pressure filled with anappropriate quantity of CFC-12, or other suitable propellant includingCFC-11, CFC-114, HFA-227, and HFA-134, in some cases approximately 4gms, to achieve an optimal phospholipid concentration range, which canbe 0.5-6 mg/mL. HFA-227 can also be used as the propellant (theoreticalphospholipid emitted dose=100 ±5 μg per spray). When using HFA-227 theformulation can contain various amounts of anhydrous ethanol (5% to 31%w/w), PEG-400 (2% to 15% w/w) and HP-β-CD (0.3% to 1.5% w/w). Suspensionphysical stability can be assessed visually and formulations withinstant coagulation-precipitation can be rejected from use. One of suchassessments is described in the following Table I:

TABLE I Formulation of HFA-227 containing Survanta ® pMDIs; emitted doseof phospholipid was 100 ± 5 μg/spray. Precipitation/coagulation can beassessed visually. Large number of ‘+’ depicts instant precipitation andin some cases sticking to the walls of the container. Six formulationsselected for aerodynamic particle sizing were Formulation Nos., 3, 4, 5,7, 8 and 11 (shaded cells).

*Theoretical vapor pressures can be calculated based on vapor pressureof ethanol = 44.6 mm Hg, vapor pressure of HFA-227 = 2925 mm Hg andvapor pressure of PEG-400 = 0.01 mm Hg at 25° C.

While the above description particularly recites the use of CFC-12 andHFA-227, one of ordinary skill in the art will recognize that othersuitable propellants or carrier mediums may likewise be used withoutdeparting from the spirit and scope of the instant invention. Forexample, CFC-11, CFC-114, HFA-134 and other propellants may also beused. In addition, it is contemplated that different methods ofaerosolizing molecules may be used, such as DPIs, nebulizers, andothers.

Appropriate formulations can be selected based on practicalconsiderations of theoretical vapor pressures and sedimentation times.For example, in one embodiment of this invention six formulations wereselected with a sedimentation time of at least 3 minutes. After theformulations are selected, aerodynamic particle size can be determined.Formulations containing the desired surfactant that yield administeredparticles slightly larger than those generated by drug-containinginhalers can be further used to determine the effect of particlemigration on tissue cells such as A549 cells using the semi-automateddetection technique described below. It is also contemplated thatdifferent types of cells may be utilized to determine surfactantproperties.

The aerodynamic particle size distribution, as used herein, correspondsto the median size of the particles, either the surfactant particles orthe medication particles. It can be assessed using two differentmethods: the USP Throat Method or the “Dome” Method. In the USP throatmethod, a cascade impactor such as the eight-stage Andersen CascadeImpactor (ACI; MSP Corp., Shoreview, Minn.) can be used (see FIG. 2,204) and operated at an appropriate flow-rate, such as 28.3 L/min±5%(flow meter from Sierra Instr. Inc., Monterey, Calif.). In oneembodiment of this method, the standard USP induction port (throat) withan internal volume of 60 mL can be employed. The pMDIs prepared for thispurpose can then be spiked with a known amount of albuterol (Cipla Ltd.,India) for quantification. In vitro deposition of albuterol administeredfrom the pMDI represents that of the phospholipid used because albuterolis present as an ethanolic solution along with the phospholipid.Initially, the pMDIs can be primed by firing 3 shots to the waste. Thenext 10 shots, with an interval between shots of, for example, 30seconds, are then made to the impactor. After the last dose ofphospholipid is fired, the inhaler is removed from the impactor and thevalve stem and actuator are rinsed with the appropriate solvent, forexample 10 mL of chromatographic mobile phase. The impactor and throatare disassembled and each plate and filter rinsed in a similar mannerwith 10 mL of the solvent. Albuterol concentrations are quantified byhigh performance liquid chromatography (HPLC) as described below. EachpMDI particle size determination can be carried out in triplicate or asmany times as needed to obtain a reliable determination. If the USPthroat method reveals high throat deposition of the selectedformulations and thus mass of particle deposits on the upper stages of 0to 2 (cut-off diameter: 9 μm to 4.7 μm) can not be assessed, the “Dome”method may be used as described below.

The “Dome” method, as shown in FIG. 2, is a modified aerodynamic sizingmethod that eliminates the USP throat and replaces it with a round flask(“dome”) 201, which may have a capacity of approximately 15 L, or othercapacities which allow for the quantification of particles. Mass of drugdeposited on each stage of the impactor 204 can be determined using theprevious method. The “dome” method can be validated by performingdeposition studies with a commercially available marketed formulation ofalbuterol (Ventolin®-HFA; GlaxoSmithKline, Research Triangle Park, N.C.)and comparing the aerodynamic distribution profiles with that obtainedusing the USP throat method.

Albuterol, deposited on the ACI stages, may be quantified using HPLC(Hitachi Ltd., Tokyo, Japan). The chromatographic separation may beachieved on a number of columns, for example the Spherex C-18, 250×4.6mm column (Phenomenex, Torrance, Calif.) with UV detection at 276 nm.The mobile phase may consist of deionized distilled water (58% v/v),acetonitrile (40% v/v), glacial acetic acid (2% v/v) and heptanesulfonic acid sodium (0.065% w/v) operated at a flow rate of 1.0 mL/minor other appropriate concentrations of mobile phase components thatfacilitate the elution and quantification of the surfactant. Using thismethod, Albuterol elutes at 3.6 mins.

A particularly preferred embodiment of the present invention comprisesthe identification of molecules to be used in the pMDI to push themedication deeper into the lungs. For this purpose an in vitro techniqueto visualize and monitor migration of latex beads placed on tissue cellsafter application of the appropriate surfactant may be used. As shown inFIG. 3, commercially available coverslips containing microgrids 301 havebeen used, for example, in locating and re-finding individual cells orcell groups, determination of cell division indices, measurement of cellgrowth and cytotaxis and estimation of cell numbers. K. Fumoto, T.Uchimura, T. Iwasaki, K. Ueda & H. Hosoya, Phosphorylation of Myosin IIRegulatory Light Chain is Necessary for Migration of HeLa Cells but notfor Localization of Myosin II at the Leading Edge, 370 Biochem. J. 551(2003); S. L. Schwindling, M. Faust & M. Montenarh, Determination ofMitotic Index by Microinjection of Fluorescently Labeled Tubulin, 81 J.Cell Biol. 169 (2002). One such coverslip microgrid is the CELLocate®gridded coverslip shown in FIG. 3. The microgrid coverslips can be usedto monitor and quantify bead movement on cell surfaces reflectingsimilar characteristics to tracheal and pulmonary tissues. For example,A549 cells may be cultured in sterile growth media containing DMEM, 10%FBS and 1% penicillin-streptomycin at 37° C. and 5% CO₂, or otherappropriate growth media conducive to regular development of the cells.A portion of the cells is added onto the gridded coverslips 301 andincubated as explained previously until confluence is established(˜48hours). Latex beads (4.5±0.28 μm in diameter) is then be applieduniformly to the coverslip shown in FIG. 3 using a thin circular ring,and the number of beads in each position can be counted using opticalmicroscopy, such as the Nikon E800, Nikon Instr. Inc., Melville, N.Y.,at 400× magnification. After the beads are placed on the coverslip 301,the desired surfactant can be applied utilizing the modified V-shaped401 adaptor shown on FIG. 4.

A rapid method of analysis can be employed to determine the amount ofphospholipids passing through the slit of the V-shaped adaptor shown inFIG. 4 and depositing on the coverslip based on complex formationbetween ammonium ferrothiocyanate and phospholipids. J. C. M Stewart,Colorimetric Determination of Phospholipids with AmmoniumFerrothiocyanate, 104 Anal. Biochem. 10 (1980). A standard solution ofammonium ferrothiocyanate can be prepared by dissolving 27.03 gms offerric chloride hexahydrate and 30.4 gms of ammonium thiocyanate in 1 Lof deionized distilled water. A solution of 10 mg of PTC in 100 mLchloroform may be prepared. Triplicate volumes of this solution, between0.1 and 2 mL, may be removed to set-up a calibration plot, added to 2 mLammonium ferrothiocyanate solution in a test tube and enough chloroformto make the final volume of 2 mL. The biphasic mixture is vortexed for30 seconds and after separation, absorbance of the lower chloroformphase is measured at λ_(max)=464 nm (Genesys2 UV Spectrophotometer;Spectronic Instr., Rochester, N.Y.). This method allows measurements ofphospholipids in the range of 10 μg to 200 μg.

pMDI-delivered phospholipids passing through the slit are dissolved in 2mL chloroform and added to 2 mL ammonium ferrothiocyanate solution. Themixture is vortexed, absorbance of the chloroform layer measured andphospholipid amount quantified based on the linear calibration plot. Theanalysis can also be employed to determine mass balance of actuatedphospholipids for ACI quantification and to validate the assumption thatalbuterol deposition is an appropriate measure of phospholipidaerodynamic distribution.

Phosphatidylcholine (PTC) represents at least 60% of both endogenous andexogenous surfactants by weight and can be used as the model surfactantto study particle movement and establish cell models. Any number ofphospholipids can be used as surfactants to drive medications deeperinto the lungs. One example of such phospholipids is a natural exogenoussurfactant, Survanta® (beractant), that can be used for achieving thedesired result. Surfactant (10-15μL of PTC, Survanta®, or any othertarget surfactant) may be added as a narrow band to one end of the grid304 utilizing the V-shaped 401 apparatus shown on FIG. 4. The movementof latex beads may then be monitored by counting the beads before addingthe surfactant and then again after allowing the surfactant to spreadfor a period of time (for example 10 seconds). Sterile water can be usedas a control.

The above mentioned surfactant effect mechanism can be automated.Automation allows one to determine the effect of the chosen surfactant,e.g. PTC or Survanta®, formulation variables on particle migration andreduce variables associated with the manual microgrid counting system.The semi-automated detection system can comprise an inverted opticalmicroscopic system (e.g., Nikon TE-2000 with image acquisition software,Nikon Instr. Inc., Melville, N.Y.) with an automated stage controllersystem (e.g., Bioprecision XY-stage, Ludl Electronic Products Ltd.,Hawthorne, N.Y.). The effect of pMDI-delivered surfactant, e.g., PTC,Survanta®, on latex bead movement can be determined using the above cellmodel. As shown in FIGS. 3 and 4, surfactant is deposited in a narrowband 304 at only one edge of an ordinary coverslip 410 by spraying eachpMDI formulation through the V-shaped adaptor 401 with a narrow slit 407at the apex. FIG. 5 shows an example of the results that can be obtainedafter using this procedure. Beads in 220 μm² virtual square grids alonga 220×11,000 μm lane (50 grids in total), predefined using the automatedstage controller coupled to the microscope, can be counted andquantified, before (initial) and after (final) application ofsurfactant. Bead migration along an extended lane can then be monitored.

On completion of each use of the system described above, the coverslipscan be treated, for example, with 25 μL of trypan blue (0.5%) andvisualized under the microscope to check for cell viability. Othermethods for determining cell viability may be used. Viable cells, giventheir intact plasma membranes, exclude the trypan blue stain whereasnonviable, membrane-permeable cells, stain dark blue. As a positivecontrol for trypan blue uptake, prior to adding trypan blue solution,cells grown on a coverslip can be incubated in a solution of 0.2% TritonX-100 in phosphate buffer saline (pH˜7.4) for 1-2 mins and the abovestaining procedure can be performed.

In one example of the application of a particular aspect of thisinvention, the latex beads (4.5±0.28 μm in diameter) can be shown tosignificantly migrate on the A549 luminal cell surface upon addition ofPTC—away from the point of application (p<0.05, ANOVA). Beads in thecell counting lane can be quantified before (Initial position) and after(Final position) application of surfactant or control, and plotted as aratio of Final:Initial counts, at each distance from the point ofsurfactant application as shown on FIG. 6. The CELLocate® microgridcounting system can be used, and fewer beads can be observed in“location 7” 307 (near which the surfactant was applied) and highernumbers can be seen in “location A” 310 (furthest observable distancefrom the surfactant application point). From the plot, these locationscan represent regions near the 110 μm and 1650 μm grid lengthrespectively.

The technique can be repeated using other surfactants such as Survanta®as shown on FIG. 4. The use of Survanta® at an original concentration of25 mg/mL of phospholipids can be shown to result in significantmigration of beads across the counting lane. The appropriate surfactantsuch as Survanta® may be pressure filled into pMDIs containing CFC-12,or other appropriate pressurizing agent, and ethanol as the co-solvent.Upon application of pMDI-formulated Survanta® (containing CFC-12 as thepropellant) using the V-shaped adaptor 401, a similar migration ofparticles can be observed. The semi-automated detection technique can beemployed to quantify particle migration across the cell surface asdescribed earlier. Virtual square grids of 220 μm² along a 220×11,000 μmlane (50 grids in total), or other distribution of grids, can bepredefined using the automated stage controller coupled to themicroscope. Beads can be counted and quantified before (Initial) andafter (Final) application of the desired surfactant. To reduce grid togrid variability in counts (noise) and summarize data more efficiently,the grids from the semi-automated counting system may be accumulatedinto groups. In some cases it may be appropriate to create groups of tengrids (representing 220×2,200 μm segments of the counting lane) andplotted as a function of bead count ratio (Final bead count insegment/Initial bead count in same segment), as shown in FIG. 7. Thisplot demonstrates how bead migration across the cell layer is dependentupon the dose of surfactant emitted by the pMDI. Significant beadmovement may be observed using pMDIs delivering different concentrationsof phospholipids. In one embodiment of this invention such concentrationwas equal to 221 μg of phospholipids (from an pMDI containing 1.8 mg/mLphospholipid, p<0.05).

The amount of phospholipid actually passing through the slit 407 of theV-shaped adaptor 401 and depositing on the coverslip 410 can bequantified based on the colorimetric assay as explained previously. Redinorganic ammonium ferrothiocyanate is insoluble in chloroform, butforms a colored complex with phospholipids, which are freely soluble inchloroform and can be quantified. The composition of this complex hasbeen determined to be phospholipid:Fe(SCN)₃::1:1. J. C. M Stewart,Colorimetric Determination of Phospholipids with AmmoniumFerrothiocyanate, 104 Anal. Biochem. 10 (1980). In one embodiment of theinvention, in which pMDI contains 1.3 mg/mL phospholipid, the amount ofphospholipid deposited can be calculated to be 16±3.2 μg/spray; and forpMDI containing 1.8 mg/mL phospholipid, the deposited amount can be27±0.8 μg/spray.

Due to practical considerations of non-ozone depleting HFA replacing theozone depleting CFC propellants in pMDIs, a preferred embodiment of theinvention utilizes ozone friendly HFA pMDIs. One example of such ozonefriendly pMDIs contains HFA-227 as the propellant of choice forreformulating the phospholipid chaser pMDIs. The dielectric constant forHFA-227 at 25° C. is 3.94. A. H. Pischtiak, M. Pittroff, & T. Schwarze,Characteristics, Supply and Use of the Hydroflurocarbon HFA 227 and HFA134a for Medical Aerosols in Past, Present and Future-ManufacturersPerspectives,www.solvay-fluor.com/docroot/fluor/static_files/attachments/characteristics.pdf(accessed Jun. 26, 2006). HFA-227 is, therefore, a slightly polarsubstance, a property that can be utilized to determine the dissolvingbehavior of HFA with the desired surfactants.

It is contemplated that the concentration of phospholipid may rangebetween 0.001 mg and 50 mg per spray, and preferably between 0.01 mg and10 mg per spray. For example, in a preferred embodiment of thisinvention CFC-12 containing pMDIs with an effective emitted dose ofphospholipid that produced significant particle migration can be foundto range within 0.082 mg to 0.111 mg per spray. pMDIs can also bepressure filled with HFA-227 and other excipients to achieve atheoretical emitted dose of phospholipid equal to, for example,0.100±0.005 mg per spray.

Anhydrous ethanol can be employed as the co-solvent to enhance thesuspension stability of the pMDIs. Increasing concentrations from 5% w/wto 31% w/w of ethanol can enhance the physical stability and reducedcoagulation and precipitation of phospholipids. At concentrations below10% w/w of ethanol, secondary stabilizing agents such as PEG-400 orHP-β-CD can be used. Both HP-β-CD and PEG are well-establishedpharmaceutical excipients and are approved for intravenousadministration. H. Steckel & S. Wehle, A Novel Formulation Technique for

Metered Dose Inhaler (pMDI) Suspensions, 284 Int. J. Pharm. 75 (2004).

In a preferred embodiment of the invention the ACI with the USP throatand the “dome” method for the selected formulations (with albuterol asthe marker for phospholipid deposition) can be used and the results canbe summarized as shown in Table II. For the USP throat method, the tablecan represent absolute mass and percent of drug deposited on theactuator, throat and different stages of the ACI. For the “dome” method,the measure of deposition can be based on mass and percent deposited onthe actuator, induction cone and stages of the ACI. From the table andthe deposition analyses, it can be shown that different ethanol amountsin the formulation and addition of non-volatile PEG-400 can increase theadministered particle size leading to the deposition of droplets higherup on the cascade impactor. For the USP throat method, due to the throatdesign and high exit velocity from the pMDI, a higher mass of particledeposition in the throat can be observed. The “dome” method can be usedto distinguish between various pMDI formulations and quantify depositionfor the region of interest in terms of molecule size (Stage 0 to Stage2: 9 to 4.7 μm cut-off diameters). These results can be verifiedassaying a commercially available marketed formulation of Ventolin®-HFAby both the USP throat and “dome” methods and observing whether theproposed “dome” method can successfully predict in vitro deposition. Inone embodiment of the present invention, the “dome” method, as shown inTable II, can successfully screen particles similar to the USP throatmethod and can also be advantageous in aiding complete dropletevaporation.

One preferred embodiment of this invention can be used to describe theeffect of formulation variables on phospholipid deposition. The impactordata can be grouped based on the method selected. The throat method candetect smaller administered particle sizes with considerable ease andthe “dome” method can identify the larger administered particles. Basedon those principles, the following stage grouping can be used:

For USP throat, mass or percent deposited: Stage3+Stage4+Stage5+Stage630Stage7+Filter

For “dome” method mass or percent deposited: Cone+Stage0+Stage1+Stage2

The data from the above grouping can be summarized as shown in Table II.From such grouping analysis, it can be shown that ethanol concentrationscan be utilized to affect particle size distribution. In one embodimentof the invention, two formulations can be selected containingapproximately 20% and 10% w/w of ethanol as the excipient and particlemigration experiments can be performed on A549 cells to determine theefficacy of these formulations.

The selected pMDI-formulations can be applied to A549 cells using theV-shaped 401 adaptor and migration of latex beads can be monitored asbefore. For these formulations containing 20% or 10% w/w ethanol(Survanta® concentration: 1.5 mg/mL), the amount of phospholipidactually deposited after exiting the slit can be determined to be 12±0.0and 17±1.2 μg/spray respectively. Significant and similar particlemigration can be observed for both these pMDI-formulations as shown inFIG. 8 (p<0.05). One skilled in the art understands that other tissuecells may be used.

Particle and device engineering approaches currently available employconsiderable modifications of conventional aerosol formulations with theultimate aim of generating small, slow moving particles with favorableaerodynamic properties and coordinating their release with the onset ofappropriate patient inhalation. Despite such complex modifications, thehigh oropharyngeal deposition of drug particles cannot be avoided. Thepresent invention takes advantage of an expanding surfactant layer,using a low cost, patient friendly, deep lung delivery technique. Thepresent invention provides that particles can effectively be “pushed”away from the expanding surfactant layer. Spreading of localizedsurfactant monolayers by surface tension gradients has recently beenmodeled mathematically. F. F. Espinosa & R. D. Kamm, Bolus DispersalThrough Lungs in Surfactant Replacement Therapy, 86 J. Appl. Physiol.391 (1999). A solid particle placed inside a fluid with a chemicalgradient can move along the direction of the gradient. A. Mikhailov & D.Meinköhn, Stochastic Dynamics, Self-Motion in Physico-Chemical SystemsFar From Thermal Equilibrium 334-345 (L. Schimansky-Geier & T. Pöscheleds., Springer, Berlin 1997). This effect takes place because thechemical substance influences the local surface tension coefficient andthus changes the intensity of surface forces applied at the liquid-solidinterface. Because of this gradient in the chemical concentration, thesurface tension forces acting on the particle are not balanced and theparticle moves along the direction of change of the chemicalconcentration. The chemical gradient can be produced by a surface activeagent and movement of particles away from an expanding surfactant layerwill be persistent due to an asymmetrical force gradient.

The measurements of surface tension due to the administeredphospholipids can be conducted using the Du Noüy Tensiometer at 25° C.The force required to detach a platinum ring from the surface of HBSS(pH˜7.4, surface tension 68.41±0.71 mN/m) in a petri dish can bemeasured for various surface concentrations of surfactant administeredfrom the pMDIs and plotted as shown in FIG. 9. Surface tension can beshown to drop based on the surface concentration of surfactant, whereone such concentration can be 11 μg/cm² (p<0.05) corresponding to 221 μgof administered phospholipids. This result can be correlated withparticle migration data and thus movement of particles on the surfacecan be defined as a surface tension mediated mechanism.

In a practical scenario, such migration deeper in the lungs can occurwhen the aerodynamic size of deposited particles containing thephospholipids are larger than those of the previously deposited drugparticles. Hence, formulations of HFA-227, for example, containing theappropriate surfactant can be developed to achieve a size slightlylarger than those of the target drug.

In a broader perspective, the following dosing strategy is envisioned:(a) the patient inhales their medication from any type of FDA approvedinhaler and the drug particles deposit on the luminal surface at alocation which depends on the inhaler characteristics and patienttechnique; and (b) the patient immediately inhales a dose of thesurfactant from the surfactant inhaler, such as a pMDI, containingparticles or droplets larger than the drug particles or droplets. Thelarger particles or droplets deposit higher in the airways and then“push” drug particles deeper into the lung.

The novel phospholipid-based inhalation system of the present inventionincreases bioavailability of aerosolized drugs, by increasing thefraction of drug that reaches its target in the bronchioles and alveolarspaces of the lung. This approach, which is based upon fundamentaltheories of surface tension reduction and phenomena of surfactantspreading, requires no modification of existing drug containing inhalersbut envisions the use of a second “chaser” inhaler. The “chaser” inhaleremploys a mature technology to deliver surfactants with a proven historyof safe use, and it is envisioned by the inventors herein that it may beuseable in conjunction with all inhaled products.

TABLE II ACI studies with selected formulations and Ventolin HFA:comparison between the USP throat method and ‘dome’ method. ‘Sum’ forthroat studies = S3 + S4 + S5 + S6 + S7 + F; ‘Sum’ for ‘dome’ studies =C + S0 + S1 + S2. Data represent mean of triplicate experiments. Valuesshow percent deposited on each stage. (Values in parentheses representmass (μg) of drug deposited). Form. Ethanol A + S T + C Stage 0 Stage 1Stage 2 Stage 3 Stage 4 Stage 5 Stage 6 Stage 7 Filter Sum #3 31.5  8.0987.7 0.66 0.15 0.22 0.71 1.01 1.01 0.35 0.11 0 (0) 3.20 (Throat) % w/w(84.6) (918) (6.91) (1.54) (2.28) (7.41) (10.6) (10.6) (3.64) (1.12)(33.3) ‘Dome’ 11.6 12.3 24.4 17.6 12.4 11.0 6.21 2.65 1.15 0.75 0 (0)66.7 (30.6) (33.6) (71.3) (51.9) (36.8) (32.5) (18.4) (7.94) (3.35)(2.12) (194) #4 20.8  11.4 79.5 1.93 0.44 0.46 1.33 1.93 1.91 0.80 0.280 (0) 6.25 (Throat) % w/w (91.9) (641) (15.6) (3.57) (3.69) (10.7)(15.6) (15.4) (6.47) (2.23) (50.4) ‘Dome’ 8.32 6.53 12.1 16.7 14.7 21.212.1 5.23 2.13 1.02 0 (0) 50.0 (29.0) (23.3) (44.0) (61.0) (53.1) (75.4)(42.8) (18.4) (7.59) (3.57) (181) #5  9.36 13.7 71.8 1.55 0.45 0.54 1.803.47 4.19 1.85 0.68 0 (0) 12.0 (Throat) % w/w (61.8) (325) (6.98) (2.01)(2.44) (8.16) (15.7) (19.0) (8.37) (3.09) (54.3) ‘Dome’ 6.68 5.01 2.032.89 6.11 20.6 28.2 20.7 5.69 2.07 0 (0) 16.0 (21.8) (16.1) (6.56)(9.33) (19.7) (66.8) (92.5) (68.4) (18.8) (6.83) (51.7) #7  6.56 14.665.0 1.55 0.52 0.49 1.87 5.03 6.64 3.10 1.16 0 (0) 17.8 (Throat) % w/w(35.9) (160) (3.86) (1.15) (1.11) (4.72) (12.4) (16.5) (7.68) (2.86)(44.1) ‘Dome’ 8.73 4.99 1.41 2.22 4.43 17.2 28.2 18.9 10.3 3.67 0 (0)13.0 (16.2) (9.17) (2.60) (4.10) (8.22) (31.9) (52.3) (35.1) (19.1)(6.83) (24.1) #8  7.87 11.1 85.9 0.96 0.53 0.42 0.51 0.37 0.25 0 (0) 0(0) 0 (0) 1.12 (Throat) % w/w (58.6) (441) (5.03) (2.74) (2.12) (2.58)(1.86) (1.22) (5.66) ‘Dome’ 35.3 7.18 26.0 12.6 5.12 6.58 3.46 2.91 0.900 (0) 0 (0) 50.8 (19.4) (3.86) (14.3) (6.97) (2.83) (3.64) (1.89) (1.58)(0.53) (28.0) #11  4.57 9.06 85.0 1.26 0.91 0.80 0.93 0.83 1.22 0 (0) 0(0) 0 (0) 2.98 (Throat) % w/w (23.8) (223) (3.31) (2.38) (2.10) (2.43)(2.18) (3.18) (7.79) ‘Dome’ 30.8 7.45 25.4 14.1 6.19 6.71 4.91 4.42 0(0) 0 (0) 0 (0) 53.1 (14.5) (3.52) (12.1) (6.70) (2.94) (3.19) (2.34)(2.09) (25.2) Vent. N/A 13.4 53.2 0.74 0.97 2.19 9.24 13.0 6.43 0.630.21 0 (0) 29.5 (Throat) (213) (849) (11.8) (15.6) (35.0) (147) (206)(101) (9.99) (3.25) (468) ‘Dome’ 11.6 3.79 1.39 2.62 7.01 29.5 32.2 10.70.87 0.30 0 (0) 14.8 (117) (37.1) (14.1) (26.9) (71.7) (299) (320) (106)(8.64) (2.91) (150)

1. A method for delivering medications to bronchioles and alveolipulmonary targets comprising: a first step of administering aerosolizedmedication into a patient's respiratory tract by a medication canistercomprising a medication, wherein said medication canister is configuredto deliver the medication in a medication spray having a medicationparticle size distribution; and a second step of administering anaerosolized surfactant formulation into the patient's respiratory tractby a surfactant canister comprising a surfactant formulation, whereinsaid surfactant canister is configured to deliver the surfactantformulation in a surfactant spray having a surfactant particle sizedistribution that is larger than the medication particle sizedistribution.
 2. The method of claim 1, wherein the surfactantaerodynamic particle size distribution is greater than 5 μm.
 3. Themethod of claim 1, wherein the surfactant formulation has a phospholipidconcentration between 0.001 mg and 50 mg per spray.
 4. The method ofclaim 4, wherein the surfactant formulation has a phospholipidconcentration between 0.01 mg and 10 mg per spray.
 5. The method ofclaim 1, wherein the medication canister and the surfactant canistercomprise a propellant.
 6. The method of claim 5, wherein the propellantis selected from the group consisting of CFC-11, CFC-12, CFC-114,HFA-134, and HFA-227.
 7. The method of claim 6, wherein the surfactantformulation has a phospholipid concentration of 82-111 μg per spray andthe propellant is CFC-12.
 8. The method of claim 7, wherein thesurfactant formulation has a phospholipid concentration of 95-105 μg perspray and the propellant is HFA-227.